Cyclase-Associated Protein is Essential for theFunctioning of the Endo-Lysosomal System andProvides a Link to the Actin Cytoskeleton

Hameeda Sultana1, Francisco Rivero1,Rosemarie Blau-Wasser1, Stephan Schwager2,Alessandra Balbo3, Salvatore Bozzaro3, MichaelSchleicher2 and Angelika A. Noegel1,*

1Center for Biochemistry and Center for MolecularMedicine Cologne, Medical Faculty, University of Cologne,50931 Koln, Germany2Institute of Cell Biology, Ludwig-Maximilians-Universitat,80336 Munchen, Germany3Dipartimento di Scienze Cliniche e Biologiche, OspedaleS. Luigi, 10043 Orbassano, Italy*Corresponding author: Dr Angelika A. Noegel,noegel@uni-koeln.de

Data from mutant analysis in yeast and Dictyosteliumindicate a role for the cyclase-associated protein (CAP)in endocytosis and vesicle transport. We have usedgenetic and biochemical approaches to identify novelinteracting partners of Dictyostelium CAP to help explainits molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunitsof the highly conserved vacuolar H+-ATPase (V-ATPase)and co-localizes to some extent with the V-ATPase.Furthermore, CAP is essential for maintaining the struc-tural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology ofV-ATPase- and Nramp1-decorated membranes were dis-turbed in a CAP mutant (CAP bsr) accompanied by anincreased endosomal pH. Moreover, concanamycin A(CMA), a specific inhibitor of the V-ATPase, had a moresevere effect on CAP bsr than on wild-type cells, and themutant did not show adaptation to the drug. Also, thedistribution of green fluorescent protein-CAP wasaffected upon CMA treatment in the wildtype and recov-ered after adaptation. Distribution of the V-ATPase inCAP bsr was drastically altered upon hypo-osmoticshock, and growth was slower and reached lower satura-tion densities in the mutant under hyper-osmotic condi-tions. Taken together, our data unravel a link of CAP withthe actin cytoskeleton and endocytosis and suggest thatCAP is an essential component of the endo-lysosomalsystem in Dictyostelium.

Key words: CAP, contractile vacuole system, F-actin-depolymerizing drugs, Nramp1, V-ATPase, V-ATPaseinhibitor

Received 14 January 2005, revised and accepted for pub-lication 8 July 2005, published on-line 8 August 2005

It has been increasingly realized that rearrangement of the

actin cytoskeleton is essential for the process of endocy-

tosis. Localized recruitment and polymerization of actin

are observed at the sites of endocytosis, and cytoskeletal

components are shown to assemble and help in localizing

the endocytic machinery to domains of the plasma mem-

brane (1). Genetic studies in yeast have revealed many

genes required for receptor-mediated endocytosis such as

Sla1p/end3, Sla2p/end4/Mop2, Abp1p, Rvs167p, Srv2p/

CAP (cyclase-associated protein), Pan1, Arc15, Act1p and

Aip1p, which regulate actin dynamics and act as bridges or

adapters between the actin cytoskeleton and the endocy-

tic machinery (2–4). These proteins assemble into modular

complexes that can induce actin polymerization at the

sites of endocytosis. In elegant studies, Kaksonen et al.

(5) have unraveled a finely choreographed pathway of the

assembly of cortical patches of differing protein composi-

tion at the plasma membrane during endocytic internaliza-

tion and have shown that actin and Sla2p are directly

involved in the internalization and are required for patch

motility. Sla2p is a protein functioning at the interface

between the actin cytoskeleton and the endocytic machin-

ery and is suggested to link actin polymerization and endo-

cytic internalization based on findings that in sla2D cells

patch motility is blocked and actin remains at the cell

cortex. Furthermore, Sla2p is a candidate for negatively

regulating the Arp2/3 complex-mediated actin nucleation

as endocytic sites in sla2D cells had more cortical F-actin

than the wildtype.

The mammalian homologs of the yeast patch proteins

Sla2p and Pan1p and Hip1R and Eps15, localize to

clathrin-coated pits (CCPs), the sites of endocytic interna-

lization.Hip1R isbothacomponentofCCPsandclathrin-coated

vesicles (CCVs) and functions in the receptor-mediated endo-

cytosis similar to Sla2p. Hip1R is associated with clathrin

during CCVs’ formation and frequently localizes to the

edges of the forming coated pits, similar to cortactin,

and is suggested to connect clathrin to F-actin at the

cortex. Furthermore, Hip1R binds to cortactin and can

physically connect actin and clathrin in vitro (6). The deple-

tion of Hip1R did not disrupt the transient association

between endocytic and cytoskeletal proteins but rather

stabilized it, whereas RNAi double-depletion experiments

for Hip1R and cortactin demonstrated that accumulation

of the cortical actin–endocytic complexes depended on

cortactin (7).

Traffic 2005; 6: 930–946Copyright # Blackwell Munksgaard 2005

Blackwell Munksgaard doi: 10.1111/j.1600-0854.2005.00330.x

930

The other candidates that may link the actin cytoskeleton

to endocytosis are Abp1p, Rvs167p and Srv2p. They co-

localize with the cortical actin cytoskeleton and relocalize

to sites of cytoskeletal rearrangements. More recent stu-

dies of the mammalian Abp1p have strengthened this role

by demonstrating an interaction with dynamin, a large

GTPase controlling the fission reaction during endocytosis

(8). Furthermore, amphiphysins, the homologs of yeast

Rvs167p, also interact with dynamin to regulate endocy-

tosis of synaptic vesicles (9).

Srv2p/cyclase-associated protein, a further component

of the endocytic machinery and an evolutionarily

conserved regulator of the G-actin/F-actin ratio, is sug-

gested to provide a link to the dynamin–Abp1p– amphy-

physin complex, because the yeast protein interacts

directly or indirectly with many key components of the

actin cytoskeleton and endocytosis like Abp1p, Sla2p

and Rvs167p (10). Recent papers provided evidence

for an active role of Srv2/CAP in controlling actin fila-

ment dynamics and showed that Srv2/CAP exists as a

high molecular weight structure (approximately

600 kDa) composed of actin and Srv2 (1:1 M ratio),

which is linked to actin filaments via the SH3 domain

of Abp1 (11). This Srv2–actin complex functions as a

monomer-processing intermediate, which catalytically

accelerates cofilin-dependent actin turnover by releasing

cofilin from ADP-G-actin monomers allowing recycling

of cofilin for new rounds of filament depolymerization.

The nucleotide exchange on ADP-G-actin is enhanced,

and due to the lower affinity of Srv2/CAP for ATP-G-

actin, other cellular factors, such as profilin or WASP,

may take over ATP-G-actin and facilitate actin assembly

(12–15). Profilin and Srv2 were also reported to physi-

cally interact (14,16), which might further facilitate a

monomer handoff. The cofilin-induced acceleration of

actin turnover has been shown to be achieved through

the integration of the activities of CAP, which involves a

coordinated interplay between its N- and C-terminal

domains. These findings provide evidence on the phy-

siological significance of the reported interaction

between the N- and C-terminal domains within CAP

molecules (10).

Data from Dictyostelium also indicate an involvement of

CAP in areas of high actin rearrangements during cell

motility, pinocytosis and phagocytosis and in the genera-

tion of cell polarity (17–19). Here, we have made an

attempt to define and strengthen the role of CAP in linking

the actin cytoskeleton to endocytosis by identifying its

interacting partners and by further analysis of the

Dictyostelium CAP bsr mutant. In this mutant, the CAP

gene has been altered by homologous recombination in

such a way that the mutant had less than 5% of the

protein concentration in wild-type cells. The mutant had

a severe growth defect reaching saturation densities dur-

ing growth in suspension at 6 � 106 cells/mL in contrast

to the parent strain with 1.2 � 107 cells/mL, which

resulted from an endocytosis defect. When analyzing the

developmental properties of the mutant, we noted

defects in cAMP signaling, cell polarization and chemotac-

tic motility, which suggested an interaction of CAP with

adenylyl cyclase and an influence of the protein on signal-

ing pathways directly and through its function as a regula-

tory component of the cytoskeleton. All defects observed

in the mutant could be rescued by expression of a green

fluorescent protein (GFP)-tagged CAP (18,19).

We report here that CAP interacts with components of the

vacuolar Hþ-ATPase (V-ATPase). Vacuolar Hþ-ATPases are

highly conserved multisubunit enzymes composed of an

integral (transmembrane) V0 domain that consists of five

subunits (subunits a–d) serving as proton channel and a

cytosolic catalytic sector (peripheral V1 domain) composed

of eight subunits (subunits A–H) that contains the ATP-

binding site involved in ATPase activity. Vacuolar Hþ-

ATPases are present in the endomembranes of all and in

the plasma membrane of many eukaryotic cells. They are

responsible for the acidification of lysosomes, endo-

somes, the Golgi complex, and secretory vesicles, and

function in processes such as receptor-mediated endocy-

tosis, intracellular targeting of lysosomal enzymes, protein

processing and degradation, and the transport of small

molecules across the plasma membrane of various cell

types (20). In Dictyostelium discoideum, the V-ATPase is

also present in membranes of the contractile vacuole (CV),

an osmoregulatory organelle of freshwater and soil proto-

zoa which pumps water out of the cell (21).

Our data suggest that loss of CAP affects the structural

organization and integrity of V-ATPase and natural resis-

tance-associated macrophage protein (Nramp1)-decorated

endo-lysosomal membranes and alters the distribution and

morphology of the vesicular network. This occurs in a

manner probably similar to the action of actin-depolymer-

izing drugs. The endosomal pH in the mutant is altered

and is highly sensitive to concanamycin A (CMA), a spe-

cific inhibitor of the V-ATPase. Together, our studies pro-

vide an insight into the role and functioning of CAP as a

general regulator of the actin cytoskeleton and

endocytosis.

Results

Cyclase-associated protein interacts directly with the

V-ATPase in Dictyostelium

In a search for components that interact with CAP, we

performed a series of immunoprecipitation assays using

cell extracts of wild-type AX2, AX2 cells expressing GFP-

CAP and the CAP mutant CAP bsr for control and antibo-

dies specific to GFP and CAP. In the immunoprecipitates,

we repeatedly detected the B subunit (vatB, approxi-

mately 31 kDa) of the V1 peripheral complex and the d

subunit (41 kDa) of the integral membrane V0 complex of

the V-ATPase with highest mass spectrometric scores,

CAP Links Endocytosis to the Actin Cytoskeleton

Traffic 2005; 6: 930–946 931

whereas these proteins were absent in control experi-

ments performed with CAP bsr. Our immunoprecipitation

assays also revealed other elements of the endocytic

machinery associated with CAP and the V-ATPase (our

unpublished data). We have extended these findings by

carrying out immunoprecipitation with the GFP-specific

antibody, mAb K3-184-2, using lysates from AX2 cells

that expressed GFP-CAP. The immunoprecipitate was

probed for the presence of the 69-kDa vatA subunit of

the V-ATPase (Figure 1A) for which a monoclonal antibody

is available (22). The presence of CAP was confirmed with

mAb 223-445-1. In an independent yeast two-hybrid

screen using a Dictyostelium cDNA library, we identified

the H subunit (50 kDa) of the V1 peripheral complex of the

V-ATPase as interaction partner of the N-terminal domain

of CAP. Further studies revealed that V-ATPase H inter-

acted with full-length CAP, the N-domain (aa 1–215) and a

100 amino acids N-terminal stretch of CAP (aa 1–102)

narrowing down the interaction site (Figure 1B).

Subsequently, we also observed a direct interaction of

CAP with the d subunit of the V-ATPase by the yeast

two-hybrid assay. The association of CAP with the

V-ATPase complex and the physical interaction with its

subunits suggested a direct link of CAP with the early

and late endo-lysosomal system, because V-ATPase is a

component of these endo-membrane systems. Together,

our results indicate that CAP interacts directly with the

V-ATPase and may play a crucial role during endocytosis

and vesicular trafficking.

Cyclase-associated protein associates and co-localizes

with vacuolar membranes

The availability of reagents specific for several V-ATPase

components allowed us to investigate this interaction of

CAP by indirect immunofluorescence. AX2 cells expres-

sing GFP-CAP were immunostained for vatA, a marker for

the endo-lysosomal system and the CV (22). Green fluores-

cent protein-cyclase-associated protein is present near the

plasma membrane and is diffusely distributed in the cyto-

sol with enrichments on vesicles and vacuoles, which are

stained by the vatA antibodies as well, although not all

GFP-CAP-decorated vesicles are stained by the vatA anti-

bodies. Conversely, not all vatA positive structures carry

GFP-CAP (Figure 2A). In further studies, we also observed

a co-localization of CAP with the vatB subunit, which was

expressed as GFP fusion protein in AX2 cells, whereas

CAP was detected by a mAb. However, in this case, the

co-localization was less prominent and the CAP antibody

did not stain intracellular vesicles as strongly as is seen

when a GFP-tagged CAP is used (Figure 2B). This could be

due to the specificity of the antibody or to the higher levels

of GFP-CAP as compared with the endogenous protein,

which might allow to observe an otherwise rather transi-

ent interaction. Green fluorescent protein – vatB was pre-

sent on endo-membranes and co-localized with the vatA

subunit (data not shown). Our previous observations that

GFP-CAP rescued all phenotypic defects in the mutant

that we had observed argues that the fusion protein

behaves like the wild-type protein and is most likely prop-

erly targeted. On the basis of our interaction studies, the

distribution and association of CAP with the vesicular

membranes could be due to the direct physical interaction

of CAP with subunits of the V-ATPase.

Cyclase-associated protein is indispensable for

maintaining the integrity and organization of the

V-ATPase-decorated endo-membrane system

CAP bsr mutant cells have a severe growth and endo-

cytosis defect (18), but a link to the endosomal system

has not been tested so far. Here, we investigated the

distribution and integrity of the V-ATPase system in CAP

bsr cells with the vatA-specific mAb. In AX2 cells, vatA is

found at the periphery of large and small vesicles and also

present in the cytosol, whereas in CAP bsr cells the

structure and shape of the vatA labeled membranes

were strongly affected. We observed a reduced staining

of vatA as compared with the wildtype and many

vesicles of smaller sizes, which were present throughout

kDa

67

N-CAP

A B

CAP 300

Pro-C-CAP

fl-CAP

pAS2-143

Figure 1: Cyclase-associated protein interacts directly with

the vacuolar H+-ATPase (V-ATPase). A) Immunoprecipitation

of green fluorescent protein (GFP)-cyclase-associated protein

(CAP) from AX2 cells expressing GFP-CAP was performed with

GFP-specific mAb K3-184-2, the immunoblot containing

the immunocomplexes was probed for the presence of the

V-ATPase A subunit with mAb 221-35-2. The band observed at

67 kDa corresponds to vatA and the band at approximately

50 kDa corresponds to the IgG heavy chain. B) The H-subunit of

the V-ATPase interacts with the N-domain of CAP in a yeast two-

hybrid assay. Polypeptides corresponding to various CAP domains

were linked to the Gal4-activating domain, whereas the sequence

encoding the V-ATPase H subunit (residues 33–443) was fused to

the Gal4 DNA-binding domain. The corresponding plasmids were

transformed into yeast cells, and the interactions were assessed

by the filter lift b-galactosidase assay. Growth on selective media

and blue color development due to b-galactosidase activity are

shown. Cyclase-associated protein domains: N-CAP, aa 1–215;

CAP 300, aa 1–102; Pro-C-CAP, aa 216–464; fl-CAP, 1–464 and

pAS2-1, vector control.

Sultana et al.

932 Traffic 2005; 6: 930–946

the cytosol and enriched at the cell periphery (Figure 3A,

supplementary Figure 1S available online at http://

www.traffic.dk/suppmat/6_10.asp). An analysis of the

distribution of the vesicles in the mutant at different focal

planes using confocal microscopy showed that the vatA-

stained structures do not form a regular network and do not

appear throughout the cell, whereas in AX2 we detected

vatA in all focal planes (videos 1 and 2 available online at

http://www.traffic.dk/suppmat/6_10.asp). We also found

that CAP bsr cells were more flattened (videos 3 and 4

available online at http://www.traffic.dk/suppmat/6_10.asp).

The weaker staining for vatA in the mutant was due to

reduced levels of the protein as revealed by Western blot

analysis (Figure 3B). The reduced vatA levels further sug-

gested that CAP contributes to the integrity of the V-

ATPase and thus acts as a general regulator of endocytosis.

When we introduced a plasmid allowing expression of full-

length GFP-CAP, the vatA staining and distribution of the

vacuolar network were restored to the wild-type pattern

(Figure 3C). We extended this analysis and tested the

individual domains of CAP for their capability to re-estab-

lish an intact vesicular network. In CAP bsr expressing the

GFP-tagged N-terminal domain, the vesicular network was

restored but only to a partial extent. The vesicles were

large, less numerous and strongly stained for vatA. We

also observed a co-localization of GFP-N-CAP-Pro with

vatA at these structures (Figure 3C). Furthermore, expres-

sion of the C-terminal domain as GFP–Pro-C-CAP fusion

had some positive effect on the structure of vatA-contain-

ing vesicles but only the full-length protein efficiently cor-

rected the observed alterations (Figure 3C).

Morphology and functioning of the CV system is

altered in CAP bsr

In Dictyostelium about 10% of the total cellular V-ATPase is

associated with the endo-lysosomal system, whereas the

remainder is associated with the membranes of the tubular

system of the CV (23). The CV system (CVS) consists of

ducts, cisternae and bladders and is transiently connected

to the plasma membrane by pores through which the blad-

der expels water and thus is involved in osmoregulation

(24). The presence of V-ATPase as marker on these sys-

tems suggests that both CVS and endo-lysosomal compart-

ments are connected to each other by fusion of their

membranes, and the interaction of CAP with V-ATPase

suggested their co-ordinated role in the functioning of the

CVS. To understand the osmoregulatory role of the V-

ATPase in the absence of CAP, we performed studies

under hypo-osmotic stress conditions, as the V-ATPase-

rich CVS rapidly collects and expels water. Phase-contrast

microscopy revealed that most of the wild-type cells were

attached to the surface and exhibited an amoeboid shape

and morphology. Under hypo-osmotic conditions, wild-type

CVSs were visualized as phase-lucent compartments

(Figure 4, arrowheads) that gradually filled and swiftly con-

tracted. After successful discharge (shown by arrow), addi-

tional phase-lucent vacuoles were filled to repeat the cycle.

The mutant cells were attached and appeared to be mor-

phologically normal in growth medium; however, in water,

they rounded up and detached from the plastic surface.

CAP bsr cells hardly contained phase-lucent vacuoles and

further showed no size changes, refilling or discharge of

vacuoles for a period of more than 30 mins (Figure 4).

GFP-CAP

GFP-vatB CAP

vatA MergeA

B

Figure 2: Cyclase-associated protein co-localization with vacuolar H+-ATPase (V-ATPase). A) AX2 cells expressing green fluores-

cent protein (GFP)-cyclase-associated protein (CAP) were fixed and immunolabeled with V-ATPase subunit A (vatA)-specific mAb 221-35-2

followed by a Cy3-labeled secondary antibody. Green fluorescent protein-cyclase-associated protein co-localized partially with vatA-

stained membranes (arrowhead). The images were obtained using a confocal laser-scanning microscope. B) AX2 cells expressing GFP-

vatB were labeled with the CAP mAb 230-18-8 to analyze the co-distribution of CAP with GFP-vatB. Cyclase-associated protein was

enriched at the cell cortex and was also present on cytoplasmic structures. A closer analysis revealed its association with vacuolar

membranes and a partial co-localization with GFP-vatB (arrowhead). Bars, 5 mm.

CAP Links Endocytosis to the Actin Cytoskeleton

Traffic 2005; 6: 930–946 933

After 1–2 h, 70% of the cells had lysed, suggesting that

CAP may regulate water homeostasis because the

mutants retain low level of osmoregulation.

To support our findings on the altered CVS in CAP bsr, we

performed experiments using the styryl dye FM 4-64 (25).

By time-lapse video microscopy, we followed the inter-

nalization of FM 4-64 from the plasma membrane into the

CV. The rapid and fast events of dye redistribution, CV

filling and discharge were observed by recording one

frame per second in wild-type cells. Cyclase-associated

protein bsr also exhibited these events; however, they

appeared much slower and fewer as compared with

AX2. Mostly, the mutants swelled and appeared round

and lost their typical morphology. Occasionally, the

appearance of enlarged and filled CVs at the plasma mem-

brane was observed, which appeared to slowly expel fluid

suggesting an impaired functioning of the CV (videos

A B

C

AX2 CAP bsr

GF

P-C

AP

GF

P-N

-CA

P-P

roG

FP

-Pro

-C-C

AP

vatA Merge

vatA

CAP bsr

AX2

Comitin

Figure 3: The vacuolar H+-ATPase (V-ATPase)-labeled membrane system is disturbed in CAP bsr. A) VatA-stained vacuoles and

vesicles of varying sizes are present throughout the AX2 cells. In CAP bsr, the vesicles are smaller and less numerous. Detection was

with mAb 221-35-2 followed by secondary Cy3-labeled antibody (supplementary videos 1–4 available online at http://www.traffic.dk/

suppmat/6_10.asp) correspond to this figure). B) VatA protein levels are lower in CAP bsr. Total homogenates from 2 � 105 cells of AX2

and CAP bsr were subjected to SDS – PAGE (10% acrylamide). The blot was probed with mAb 221-35-2 for detection of vatA and mAb

190-68 for labeling of comitin as loading control. C) Restoration of vacuolar organization in CAP bsr cells expressing green fluorescent

protein (GFP) fusions of CAP. Expression of GFP-CAP completely restored the vacuolar network. The disturbed vacuolar organization and

distribution was partially complemented by the expression of GFP-N-CAP-Pro and GFP-Pro-C-CAP in CAP bsr. mAb 221-35-2 labeling

revealed the presence of vesicles and a co-localization of vatA with GFP-CAP and GFP-N-CAP-Pro. Green fluorescent protein-Pro-C-CAP

distributed more diffusely and did not co-localize with vatA. Expression of CAP domains restores mostly large but less numerous vesicles

illustrating that both domains are essential for the restoration of the disturbed vacuolar network in CAP bsr. Arrowheads denote

co-localization of GFP fusions of CAP with vatA-labeled membranes. Bars, 10 mm.

Sultana et al.

934 Traffic 2005; 6: 930–946

5 and 6 available online at http://www.traffic.dk/suppmat/

6_10.asp).

As our data indicated alterations in the functioning of the

V-ATPase in CAP bsr, we analyzed its distribution under

hypo-osmotic conditions by staining the cells for vatA. In

AX2, the morphology and distribution of the V-ATPase

was different from that in CAP bsr. The V-ATPase localized

to one side of the wild-type cells; in CAP bsr, the

V-ATPase structures were present in the cell center in a

large clustered patch with few small vesicles around

(Figure 5A,B) and agreed well with the observations in

videos 5 and 6. The actin staining suggested that in AX2

actin is also enriched in those areas of the cell cortex

where the V-ATPase is found. In CAP bsr, actin is present

in some regions at the cell periphery, and the staining was

much weaker in comparison to AX2 (Figure 5C). The

altered distribution of the V-ATPase in the absence of

CAP supports the importance of CAP for this process.

We also tested the sensitivity of the mutant cells to

hyperosmotic conditions by growing them in media of

different osmotic strength and supplementing the med-

ium either with 30 mM NaCl or 115 mM sorbitol. In the

presence of 30 mM NaCl, AX2 was only marginally

affected in growth whereas the mutant reached saturation

densities, which were about 60% of untreated control

cells. Sorbitol (115 mM) affected both strains. AX2 cells

reached saturation densities that were 60% of control

cells, CAP bsr was strongly affected and reached final

densities that were only 20–30% of untreated mutant

cells. Moreover, the duplication times were significantly

increased.

Loss of CAP results in an increased endo-lysosomal

pH and slower adaptability to CMA

Because the activity of the V-ATPase affects the endo-

lysosomal pH, we measured this parameter in living AX2

and CAP bsr cells using fluorescein isothiocyanate (FITC)-

dextran as a pH probe. Growing Dictyostelium amoebae

contain acidic lumen with a pH value of 5.4–5.8 (26). We

found that the basal pH in CAP bsr was higher (pH,

6.0 � 0.2) in comparison with the wild-type cells (pH,

5.7 � 0.2). The increased basal pH in CAP bsr was statis-

tically significant (p < 0.01, n ¼ 7, Student’s t-test). This

increase suggests an involvement of CAP in maintaining

the acidic nature of the endo-lysosomal system.

To examine whether CAP is also required for the acidifica-

tion and functioning of the endo-lysosomal compartments,

AX2C

ontr

olH

ypo-

osm

otic

CAP bsr

Figure 4: CAP bsr cells are impaired

in osmoregulation. Cells were

allowed to attach to coverslips in

nutrient medium (control) or water

(hypo-osmotic condition), and phase-

contrast images were obtained after

60 min. Wild-type cells appeared fre-

quently swollen and round under

hypo-osmotic load but were still able

to crawl and change shape and

showed fast and rapid filling and dis-

charging of contractile vacuoles

(CVs). Many strong filled (arrow-

heads) and discharging vesicles

(arrow) were observed. In CAP bsr

cells, the appearance of vesicles

was drastically reduced and only a

few vesicles were observed (arrow-

head). The CVs of CAP bsr were not

able to expel water efficiently, and

the cells exhibited enlarged filled

vacuoles (arrow). Furthermore, they

barely remained attached to the cov-

erslip. Bars, 10 mm.

CAP Links Endocytosis to the Actin Cytoskeleton

Traffic 2005; 6: 930–946 935

we investigated the endosomal pH evolution following a

5-min FITC-dextran pulse in CAP bsr cells. In both AX2 and

CAP bsr, endosomal acidification was rapid, reaching a pH

value of approximately 5 during the pulse phase. During

the next 30 min, the pH value steadily rose to stabilize at

values around 6 (Figure 6A). We next investigated the

effects of CMA in CAP bsr cells. Concanamycin A is an

extremely potent inhibitor of V-ATPases and as such can

inhibit endosomal and phagosomal membrane traffic in

Dictyostelium (26). The effects of CMA (10 mM) on the

pH of endosomal and lysosomal compartments of AX2

and CAP bsr were determined by dual excitation ratio

fluorimetry of cells using FITC-dextran. To determine

how rapidly the increase in pH of the endo-lysosomes

occurs after CMA treatment, we exposed FITC-dextran-

loaded cells to CMA, and the pH was measured over the

indicated times. We observed that the rise in pH was rapid

and reached a value of approximately 6.5 at 1 h after drug

addition in both the wild-type and CAP bsr cells with no

significant difference (Figure 6B). Later on, AX2 cells

adapted to CMA and the pH dropped. Such behavior has

been reported for wild-type cells previously (26). In con-

trast, in the mutant cells, the pH increased further to a

value close to 7.0 and remained there for more than 3 h of

CMA treatment indicating that the cells did not adapt. The

increase in basal pH and the low degree of adaptability to

the effects of CMA in CAP bsr indicate that CAP is essen-

tial for the functioning of the V-ATPase.

Furthermore, to understand the complexity, dynamics and

the association of CAP with the endo-lysosomal system,

we studied the effects of CMA in vivo. The distribution of

GFP-CAP in AX2 wildtype was affected upon treatment

with CMA and showed a punctuated pattern in the cyto-

plasm. Macropinocytosis, cell shape changes and the relo-

calization of GFP-CAP to the sites of endocytosis

disappeared upon CMA treatment in comparison with the

control; however, after 60 min, these effects were re-

established upon adaptation to CMA (videos 7 and 8 avail-

able online at http://www.traffic.dk/suppmat/6_10.asp).

During endocytic transit, the V-ATPase accumulates at

phagosomes shortly after internalization occurs and is

retrieved prior to exocytosis (27). We investigated

whether absence of CAP alters the behavior of V-ATPase

during phagocytosis of yeast cells. For this, AX2 and CAP

bsr were pulsed with yeasts for 15 min followed by a

short (15 min) or long (120 min) chase period prior to

fixation (Figure 6C–F). In both strains, vatA was found

around phagosomes devoid of an actin coat. Early phago-

somes (Figure 6C,D: arrowheads) were actin-coated and

lacked vatA. Prior to exocytosis vatA detached from some

phagosomes (Figure 6E,F: asterisks). We conclude that

relocalization of V-ATPase during phagocytosis is not

noticeably impaired in CAP-bsr.

Nramp1-decorated vesicles are disturbed in CAP bsr

Dictyostelium amoebae are professional phagocytes,

which ingest bacteria as their principal food source in

both natural and laboratory conditions. They harbor a set

of proteins required for endocytosis, which are similar to

the one in higher eukaryotes. One of these proteins is the

membrane protein Nramp1 that in mammals causes resist-

ance to microbe infection with intracellular parasites such

as Mycobacteria, Salmonella and Leishmania (28). During

infection, Nramp1 is recruited to phagosomal membranes

from the late endosomal/lysosomal compartment where it

is involved in the acidification of the lumen by promoting

the fusion of the vesicles with membranes carrying

AX2

A

B

C

CAP bsr

Figure 5: The morphology and distribution of the contractile

vacuole system is disturbed in CAP bsr under hypo-osmotic

stress. A) The vatA staining showed that in AX2 cells the

V-ATPase carrying membranes localize to one side of the cells.

In contrast, CAP bsr cells had the V-ATPase-stained structures

distributed in the cytoplasm exhibiting also an altered morphology

(Bars, 5 mm). B) The corresponding phase-contrast images reveal

the cell boundaries. C) AX2 stained for actin shows the actin

cortex mostly at one side of the cells, whereas in CAP bsr, actin

was found in some peripheral regions and the actin staining

appeared reduced under the hypo-osmotic load. Bars, 10 mm.

Sultana et al.

936 Traffic 2005; 6: 930–946

6.5

A

C

D

E

*

*

F

B

6

5.5

pH pH

5

7.5

7

6.5

AX2-CMAAX2-EtOHCAP-CMACAP-EtOH

6

5.5

54.50 10 20 30

Time (min)

vatA Actin Merge Transmission

40 50

AX2CAP

60 0 30 60Time (min)

90 120 150

Figure 6: Behavior of vacuolar H+-ATPase (V-ATPase) in CAP bsr. A) Change of endosomal pH during transit of a fluid phase marker.

Cells were pulsed with fluorescein isothiocyanate (FITC)-dextran (2 mg/mL) for 10 min (dotted line), washed and resuspended in fresh

nutrient medium. Samples were withdrawn at the times indicated, and dual excitation ratio was used to calculate the endosomal pH. B)

Effects of concanamycin A (CMA) on the endosomal pH in CAP bsr. Cells were loaded with FITC-dextran (2 mg/mL) for 3 h; the specific

V-ATPase inhibitor CMA (20 mM) or its diluent ethanol (0.01% v/v) was then added, and samples were withdrawn at the times indicated for

determination of endosomal pH as described in (A). Concanamycin A inhibited acidification and increased the endo-lysosomal pH in a

significant manner. After 60 min of drug treatment, a slight recovery was gained as the pH decreased to a value of 6.0 in the wild-type

cells. In CAP bsr, the endosomal pH was near to neutral and the cells failed to adapt to CMA. Data of A and B are the average �SD of four

independent experiments. For simplicity, error bars are depicted only in one direction. (Also see supplementary videos 5 and 6 available

online at http://www.traffic.dk/suppmat/6_10.asp). C – F) Distribution of V-ATPase during phagocytosis. Cells of AX2 (C, E) and CAP bsr

(D, F) were deposited on coverslips and incubated with yeasts for 15 min, then washed and incubated in Soerensen buffer for additional

15 min (C, D) or 120 min (E, F) and fixed with picric acid/paraformaldehyde. Vacuolar Hþ-ATPase was detected with mAb 221-35-2 for

vatA followed by Cy3-labeled secondary antibody. F-actin was stained with FITC-phalloidin. The images were obtained using a confocal

laser-scanning microscope. VatA localizes around phagosomes devoid of an actin coat. Early phagosomes (arrowheads) are actin-coated

and lack vatA. In the cell shown in D, the early phagosomes are located adjacent to vatA-stained vesicles. Prior to exocytosis, vatA

detaches from the phagosomes (asterisks). V-ATPase behaves similarly in AX2 and CAP bsr. Bar, 10 mm.

CAP Links Endocytosis to the Actin Cytoskeleton

Traffic 2005; 6: 930–946 937

V-ATPase. Mammalian cells have also an Nramp2, which

is much more widely expressed in the body. Dictyostelium

discoideum harbors two homologs of Nramp, NrampA and

NrampB. They are encoded by separate genes giving rise

to 70- and 53-kDa proteins, respectively. NrampA and

NrampB (in the following designated as Nramp1) have

32% identity and 57% similarity amongst each other and

42–45% identity with the mammalian proteins.

We studied the distribution of Nramp1-GFP and found that

it is targeted to internal membranes that resemble the

vatA-stained structures. In CAP bsr, the Nramp1-GFP dis-

tribution was different from wildtype. The Nramp1-GFP-

stained membranes showed a disturbed organization, and

the stained vesicles were smaller (Figure 7A). Upon

co-staining with vatA-specific antibodies, we detected a

considerable amount of overlap; however, not all vatA-labeled

membranes carried Nramp1-GFP suggesting that Nramp1

is present on a subset of vatA-positive membranes

(Figure 7B), which are however, not identical to the CVS

(S. Bozzaro, unpublished). The altered distribution and

difference in the organization of Nramp1-GFP-

decorated membranes in AX2 and CAP bsr cells were

confirmed by live microscopy studies (videos 9 and

10 available online at http://www.traffic.dk/suppmat/

6_10.asp). Live imaging revealed that the Nramp1 vesicles

fail to undergo fast and dynamic events of movement and

fusion in the absence of CAP. The disappearance of larger

and numerous Nramp1-decorated vesicles resembles and

correlates with the situation noted for the V-ATPase. We

conclude that membranes carrying vatA, Nramp1 or both

are grossly disturbed in the mutant.

We also examined the association of CAP with the com-

partments decorated by Nramp1-GFP and stained AX2

cells expressing Nramp1-GFP with CAP-specific mAb

230-18-8 (17). This antibody strongly labels the cell cortex

and in addition localizes to some Nramp1-GFP-positive

membranes in the cytosol (Figure 8A). Furthermore, as

Nramp1 tightly associates with lysosomal-associated

membrane protein 1 (Lamp1)-positive compartments in

mammalian cells (28), we studied such an association in

A

B

AX2

GFP-Nramp1

GF

P-N

ram

p1A

X2

CA

P b

sr

vatA Merge

CAP bsr

Figure 7: Disturbance of the

endo-lysosomal system in CAP

bsr as detected by natural resist-

ance-associated macrophage

protein (Nramp1). A) AX2 and

CAP bsr cells expressing green

fluorescent protein (GFP)-Nramp1

were imaged to analyze the organi-

zation and distribution of Nramp1-

decorated vesicles. In CAP bsr the

vesicles were small, diffused and

appeared as a clustered network.

B) Organization and integrity of the

early and late endo-lysosomal sys-

tem is disturbed in CAP bsr. Early

and late endosomes are detected

using vatA staining and GFP-

Nramp1 labels late endosomes.

Mutant and wild-type cells expres-

sing GFP-Nramp1 were labeled with

mAb 221-35-2 for vatA followed by

Cy3-labeled secondary antibody. In

CAP bsr, the GFP-Nramp1 and vatA-

labeled vesicles were smaller and

vatA staining was reduced (also

see supplementary videos 7 and 8

available online at http://www.

traffic.dk/suppmat/6_10.asp).

Sultana et al.

938 Traffic 2005; 6: 930–946

Dictyostelium and found that CAP co-localizes with the

postlysosomal marker vacuolin A (Figure 8B).

Disruption of the actin cytoskeleton also disturbs the

vesicular network

Our results indicate a direct link between the actin cyto-

skeleton and membranes of the endo-lysosomal system

through CAP and subunits of the vacuolar ATPase. We

therefore tested whether the structure and integrity of the

V-ATPase system is disturbed when the actin cytoskele-

ton is disrupted using cytochalasin A (20 mM) and latruncu-

lin B (1 mM). The action of these drugs is different:

cytochalasin A binds to the barbed ends of actin filaments

inhibiting both the association and dissociation of sub-

units, whereas latrunculin B is an actin monomer seques-

tering toxin inhibiting actin polymerization and disrupting

microfilament organization as well as microfilament-

mediated processes and is 10- to 100-fold more potent

than cytochalasin (29).

We first analyzed the action of both drugs on the actin

cytoskeleton of wildtype and mutant. The treatments led

to an altered actin staining in both strains; however, CAP

bsr cells showed a different response to cytochalasin A.

After a 20 min treatment with cytochalasin A, actin was

no longer present in the cortex in AX2 cells. Instead, it

was distributed in spots all over the cells. At 40 min actin

was concentrated in large clumps inside the cells. In the

mutant, actin was still present at the cortex after 40 min

of incubation and, in addition, we observed rod-like struc-

tures in most cells. Also, the cortical staining remained

after 60 and 80 min of incubation (data not shown). In both

strains the cell shape was altered by cytochalasin A, and

cells were more rounded and did not adhere well to the

coverslips (Figure 9A). When we investigated the distribu-

tion of the vacuolar network in AX2 and CAP bsr cells

upon treatment with cytochalasin A, we observed an

altered vacuolar organization in AX2, whereas CAP bsr

cells did not show much difference in the vacuolar staining

in comparison with the control cells. In AX2 the vesicles

were fewer and small in size, and the vatA staining was

more diffused (Figure 9B).

We also found that the distribution of GFP-CAP expressed

in CAP bsr cells was affected in a way resembling the one

observed for actin upon treatment with cytochalasin A for

60 min, thus suggesting that the actin-depolymerizing

drug also affects the actin regulatory protein CAP

(Figure 10). The effect of latrunculin B was comparable

in AX2 and CAP bsr. At 10 min, the cortical staining was

less prominent and appeared fragmented, and the inten-

sity of actin staining seemed to decrease over the time

course of incubation (30 min) (Figure 11A). The vacuolar

staining was also altered in both strains. The vesicular

structures appeared to be clustered at the edges of the

cells, and the enrichment of vatA on vacuolar membranes

was less prominent upon treatment with latrunculin B

(Figure 11B). Taken together, the treatment of AX2 cells

with actin-affecting drugs had effects on the distribution

and organization of the V-ATPase that were comparable

to the ones in CAP bsr suggesting that CAP contributes

to V-ATPase localization and structural organization prob-

ably through a general role in actin organization and

dynamics.

A GFP-Nramp 1

CAP-GFP Vacuolin A

CAP Merge

MergeB

*

Figure 8: CAP co-localizes with natural resistance-associated macrophage protein (Nramp1) and vacuolin, markers of the

endo-lysosomal system. A) In AX2 cells expressing green fluorescent protein (GFP)-Nramp1, cyclase-associated protein (CAP) associ-

ates to some extent with the Nramp1-decorated vesicular endomembranes. Cyclase-associated protein was detected with mAb 230-18-

8. Arrowheads point to co-stained vesicles and star represents the association of CAP with a subset of early endosomes. B) Presence of

GFP-CAP at the postlysosomal compartment and co-localization with vacuolin A. AX2 cells expressing GFP-CAP were fixed and immuno-

stained with the postlysosomal marker vacuolin A using mAb 221-1-1. Green fluorescent protein-cyclase-associated protein was found on

some of the vacuolin A-positive vesicles (arrowheads) but did not completely overlap. Bar, 5 mm.

CAP Links Endocytosis to the Actin Cytoskeleton

Traffic 2005; 6: 930–946 939

Discussion

Cyclase-associated protein as a key component in

maintaining the organization and functioning of

V-ATPase-decorated endo-lysosomal and CV

membranes

The V-ATPases are ATP-dependent proton pumps and are

universal components of eukaryotic organisms present in

the membranes of many intracellular organelles. They

function to couple the energy of ATP hydrolysis to the

active transport of protons from the cytoplasm to the

lumen, creating a proton gradient and generating the low

intravacuolar pH found in endosomes and lysosomes (30).

The endocytic pathway defines membrane traffic from the

cell surface to the degradative compartments like lyso-

somes (animals) and vacuoles (plants and fungi), and

V-ATPases have a vital role in both endocytosis and vesicle

trafficking (20). Our attempts to identify binding partners

of CAP resulted in the isolation of various subunits of the

V-ATPase. Furthermore, we also found in an independent

yeast two-hybrid screen that the N-terminal domain of

CAP directly interacted with the H subunit of the peri-

pheral V1 complex, thus providing a direct link of CAP

with V-ATPase and establishing CAP as a component of

the endo-lysosomal membranes. The association of CAP

with the V-ATPase complex was further established by

our immunofluorescence studies where GFP-CAP co-loca-

lized with vatA-stained membranes, and endogenous CAP

overlapped with GFP-vatB to some degree (Figure 2). The

subunit B appears to be essential for the assembly of the

V-ATPase probably via its interaction with the actin cyto-

skeleton (31,32).

Several proteins have been identified that bind to subunits

of the V-ATPase and thus provide multiple links of this

enzyme to cellular components. The direct interactions

between Nef, a HIV accessory protein, and the V-ATPase

subunit H have been shown by Lu et al. (33). The binding

of Nef to the subunit H facilitates the internalization of

CD4, a primary receptor for HIV on the surface of infected

cells suggesting an important role for V-ATPase subunit H

in viral infectivity (34). Presumably, if the interaction of

CAP with the V-ATPase subunit H is conserved in humans,

this interaction may hinder the binding of the subunit H to

Nef and in turn may influence the viral infectivity.

However, this is a mere hypothesis that needs to be

thoroughly explored.

In Dictyostelium, unlike yeast, V-ATPase associates with a

variety of organelles including lysosomes and phago-

somes. Additionally, V-ATPase, calmodulin and Rab-

GTPases are present at the osmoregulatory CV complex

(25,35,36). Interestingly, in our immunoprecipitation

experiments, we found CAP in association with Rab-

GTPases (Rab4, Rab11, RabA and RabB), and in immuno-

fluorescence studies CAP co-localized with Rab-GTPases

(our unpublished results). Our findings that the functioning

of endocytic processes and the osmotic resistance are

disturbed and impaired in the CAP-deficient mutant further

provide functional support for the association and inter-

action of CAP with the V-ATPase. The sensitivity to and

lysis of CAP bsr cells in hypo-osmotic medium provided

evidence for a poor functioning of the CV system to expel

water under these stress conditions. The altered distribu-

tion of the V-ATPase under hypo-osmotic stress in the

absence of CAP further suggested the requirement of

the tight interaction of CAP with V-ATPase to achieve

the normal morphology and functioning of the proton

ControlA

X2

CA

P b

srC

AP

bsr

AX

2

A

B

Cytochalasin A

Figure 9: Cytochalasin A alters the distribution of the actin

cytoskeleton and the vacuolar network. Cells were treated

with 20 mM cytochalasin A or DMSO (control) for 40 min and

stained for actin (mAb act-1–7) (A) or vatA (mAb 221-35-2) (B). In

AX2, cytochalasin A leads to an accumulation of actin in spots and

clumps in the cytosol (A), and the vatA staining is weaker (B). In

CAP bsr cells, actin was found in the cell cortex and in rods that

centered in the cytoplasm (A). The vatA staining was less influ-

enced and did not vary much from the control (B). Bars, 10 mm.

Sultana et al.

940 Traffic 2005; 6: 930–946

pumps. Moreover, the observed reduced expression

levels of vatA in CAP bsr suggest an impaired functioning

of the V-ATPase as vatA is the subunit containing the site

of ATP hydrolysis in the enzyme. In yeast and fungi, dis-

ruption of the V-ATPase subunit A (vmaA1) inhibited nor-

mal growth, abolished sporulation and resulted in

morphological changes and reduced growth similar to

those observed after addition of CMA (37). In

Dictyostelium, reduced expression levels of VatM have

effects on cell growth and cytosolic pH regulation (38).

Our rescue analysis revealed that CAP is essential for the

structure and integrity of the endo-lysosomal membrane

network as expression of GFP-CAP led to its complete

restoration in the mutant. A partial restoration was

achieved by the expression of N- and C-domains. The

rather modest effects observed with the C-terminal

domain could be due to its association with actin or its

linkage to other components through the proline rich

region, which has the property to bind to SH3 domains

or a combination of both.

Involvement of CAP in the maintenance of endosomal

pH

The maintenance of an appropriate pH within a mem-

brane-surrounded organelle (both secretory and endo-

cytic) is a challenge from the simplest eukaryote to

complex multicellular organisms, and the balance

between active Hþ pumping and passive Hþ efflux activ-

ity of the V-ATPase achieves maintenance of an optimal

pH. The pH varies in different subcompartments of the

endocytic and secretory pathways, and the pH of each

organelle critically determines the coordinated biochem-

ical reactions. The altered structural organization of the

endo-lysosomal system correlated with the impaired

functional activity of V-ATPase, because the endosomal

pH was significantly higher in CAP bsr (Figure 6A,B).

These findings suggest an impaired function of the pro-

ton pumps to generate the proton gradient and maintain

the acidic pH of the endo-lysosomal system. There are

several lines of evidence supporting the notion that acid-

ification of the endo-lysosomes by V-ATPase is essential

for efficient targeting of molecules through the endo-

lysosomal pathway, and alterations in the normal pH

homeostasis can lead to significant functional changes

(26,39). The increase in the endosomal pH may also

account for the reduced endocytosis in CAP bsr.

Similarly, Dictyostelium mutants in distinct ABC transpor-

ters showed an altered endosomal pH and endocytosis

defects (40).

The proton pumping activity of V-ATPase is inferred

from the effects of pharmacological agents that cause

a rapid alkalinization of the acidic organelles (39). The

macrolide antibiotic bafilomycin and the related CMA

are highly specific inhibitors of V-ATPases that slow

down the receptor-mediated endocytosis and the recy-

cling of the endo-lysosomal system (30). In

Dictyostelium, CMA results in the neutralization of the

lumen of the endo-lysosomal system, inhibition of endo-

cytosis, exocytosis and phagocytosis, delays protein

processing and induces missorting of the lysosomal

enzyme a-mannosidase and causes gross morphological

changes. Concanamycin A inhibited the acidification of

endo-lysosomal vesicles and led to an increase of the

pH; however, after 60 min, an adaptation to CMA was

observed in AX2 that was associated with a decrease in

pH. Addition of fresh CMA had no effect on increasing

GFP-CAPC

ontr

olC

ytoc

hala

sin

AActin Merge

60 min

Figure 10: Cytochalasin A affects

the distribution of green fluores-

cent protein (GFP)-CAP. Cyclase-

associated protein bsr cells expres-

sing GFP-CAP were fixed and

stained to visualize actin. The distri-

bution of GFP-CAP was affected

upon treatment with the drug and

distributed in actin rich patches fail-

ing to reach the cell cortex and

showed a pattern comparable to

the actin distribution, suggesting

that the actin-depolymerizing drug

through altering the actin cytoskele-

ton also affects the CAP distribu-

tion. Bar, 10 mm.

CAP Links Endocytosis to the Actin Cytoskeleton

Traffic 2005; 6: 930–946 941

the pH, and the recovery of the pH did not require

protein synthesis. Adaptation to CMA was therefore

proposed to result from reactivation of V-ATPase func-

tion by cytosolic activators (26). In the CAP bsr mutant,

CMA caused a rapid increase in endosomal pH (nearing

a neutral pH of 7) and a failure to adapt to CMA sug-

gesting a higher sensitivity of the mutant’s V-ATPase.

The altered distribution and dynamics of GFP-CAP upon

CMA treatment further suggested an association of

CAP with the V-ATPase (videos 7 and 8 available online

at http://www.traffic.dk/suppmat/6_10.asp). It might

well be that CAP and the link to the actin cytoskeleton

provided through CAP might be identical to the ‘cyto-

solic activators’ proposed by Temesvari et al. (26).

ControlA

B

AX2

10 m

in30

min

10 m

in30

min

Latrunculin B Latrunculin BControl

CAP bsr

AX2 CAP bsr

Figure 11: Effects of latrunculin B. A) Cytoskeletal rearrangements in response to latrunculin B. AX2 and CAP bsr cells were either

treated with ethanol (control) or latrunculin B (1 mM) in phosphate buffer for the times indicated prior to fixation. Cells were labeled with

actin-specific mAb Act 1–7 followed by labeling with secondary antibody. The confocal images reveal reduction in the actin staining upon

treatment with latrunculin B in wildtype and mutant alike. B) The vacuolar network is affected during cytoskeletal changes upon treatment

with latrunculin B. Cells were treated with ethanol or latrunculin B and fixed as described in (A). The vatA labeling revealed a cytoskeleton

association of actin with the endo-lysosomal and contractile vacuole system marked by mAb 221-35-2 for vatA. In latrunculin B-treated

AX2 cells, the vatA-stained vesicles were small and clustered to one region of the cell thereby altering the vacuolar organization. Also, we

observed a weaker vacuolar staining. CAP bsr showed similar effects upon treatment with latrunculin B. Bars, 10 mm.

Sultana et al.

942 Traffic 2005; 6: 930–946

Previously, CAP has been shown to be involved in endo-

cytic internalization (18) with reduced endocytosis, while

in the current study, we have found that the vatA levels

and the vacuolar organization are affected in the absence

of CAP. Based on the role of CAP in endocytosis, it is also

formally possible that CAP regulates the V-ATPase local-

ization by contributing to the overall internalization of lipids

and proteins at the plasma membrane. In wild-type cells,

the efficient internalization of V-ATPase may lead to nor-

mal endocytosis, whereas in CAP bsr due to inefficient

internalization of the V-ATPase, it may remain at the

plasma membrane due to a general defect in endocytosis.

It is also noteworthy that the treatment of cells with actin

drugs had similar effects on the organization and distribu-

tion of the V-ATPase as the loss of CAP, which highlights a

role of CAP in facilitating and controlling the actin

dynamics that may contribute to V-ATPase localization

and distribution. However, on the basis of the data that

indicate a direct CAP–V-ATPase interaction, we assume

that the altered distribution and functioning of V-ATPase is

directly related to the loss of CAP and the interaction to

the cytoskeleton, which it provided.

Requirement of CAP for the integrity of the Nramp1-

associated membrane system

Nramp1, an integral membrane protein, is localized to the

endosomal/lysosomal compartment in macrophages and

is rapidly recruited to the membranes of the particle/

microbe-containing phagosomes upon phagocytosis (41).

Dictyostelium, a professional phagocyte principally feed-

ing on bacteria, contains two homologs of human Nramp.

Here, we have studied NrampB/Nramp1, a 53-kDa protein,

which is closer to human Nramp1 than Dictyostelium

NrampA. We have found that the Nramp1-decorated

membrane network is disturbed in CAP bsr cells suggest-

ing that CAP is involved in maintaining also this membrane

compartment. In wild-type cells, Nramp1-GFP localizes to

a subset of vatA-positive vesicles and shows some degree

of co-localization with CAP on vesicles (Figures 7 and 8).

Dictyostelium Nramp1 can therefore be classified as a

protein of the endo-lysosomal system; however, it is also

present on membranes of different origin. Our live

imaging showed that the fusion, fission and movement

events of Nramp1-GFP-decorated vesicles were much

slower in the mutant as compared with the wildtype

further supporting the disturbed V-ATPase functioning

associated with high endosomal pH (videos 9 and 10

available online at http://www.traffic.dk/suppmat/

6_10.asp).

Nramp1 tightly associates with Lamp1-positive compart-

ments and Rab7-positive late endosomal structures,

which are clearly distinct from those of the early endo-

somal marker Rab5 (28). In the endocytic pathway of

Dictyostelium, four distinct phases have been distin-

guished: the uptake of particle/fluid by phagocytosis and

macropinocytosis, an acidic phase initiated by the associa-

tion of V-ATPase with the endosomes, targeting of

lysosomal enzymes and members of the Rab family to

the V-ATPase complex and followed by a postlysosomal

period where the pH is close to neutral and vesicles are

devoid of the V-ATPase and finally exocytosis (22). The

indigestible remnants of the postlysosomal compartment

are released by exocytosis, and this compartment is

characterized by two isoforms of vacuolin, A and B,

which are encoded by different genes.

Our finding that GFP-CAP associates to some extent with

membranes carrying the lysosomal marker vacuolin A

further supported the possible connection of CAP with

Nramp1-labeled membranes (Figure 8). Acidification of

late endosomes causes the release of lysosomal enzymes

and permits receptor recycling, uncoupling of receptor–

ligand complexes, degradative processes and biosynth-

esis and sorting of lysosomal hydrolases (20,26).

Therefore, association of CAP with the lysosomal marker

and its involvement in pH homeostasis suggest a consid-

erable role of CAP in the complete endocytic circuit.

Nramp1 is initially located in the late endosomal/lysosomal

compartment and is then recruited to the phagosomes

during the course of its maturation from early plasma

membrane-derived phagosomes to phagolysosomes and

becomes enriched with the membranous compartments

containing the ingested particle. In Dictyostelium, relocal-

ization of CAP upon a stimulus during phagocytosis to the

phagocytic cups and phagosomes (our unpublished

results) and its association with Nramp1 and vacuolin A-

positive membranes may also propose a role for CAP in

phagolysosomes.

Role of CAP in linking endocytosis to the actin

cytoskeleton

Insight into the mechanism and requirement of the actin

cytoskeleton for endocytosis has come through the

identification of the genes affected in fluid phase and

receptor-mediated endocytosis with defects in cytoskeleton

organization. In addition, mutations in proteins that

regulate the integrity and modulation of the actin cytoske-

leton such as fimbrin, calmodulin and type I unconven-

tional myosin blocked endocytosis, suggesting that

mutations that disturb the integrity of the actin cytoskele-

ton affect endocytosis (1). Adessi et al. (42) proposed that

the presence of actin on Dictyostelium endocytic vesicles

reflects tight interactions between the vesicles and the

actin cytoskeleton, postulating that interactions occur via

unknown vesicle-associated actin-binding proteins. Our

data provide a link of endocytosis to the actin cytoskeleton

through CAP.

Several recent studies have suggested an essential link

between the V-ATPase to the actin cytoskeleton where

both the V1 complex and the holoenzyme are shown to

directly interact with F-actin. Interactions between

V-ATPase and microfilaments have been reported during

osteoclast activation, where ruffled membrane V-ATPases

directly bind to F-actin (31). Binding to F-actin was

CAP Links Endocytosis to the Actin Cytoskeleton

Traffic 2005; 6: 930–946 943

attributed to the amino-terminal domain of the subunit B

implicating a role for the actin–V-ATPase complex in

controlling the transport of V-ATPase to the membrane

ruffles (31,32). The interaction of CAP with the B subunit

may also provide a direct link of V-ATPase to the actin

regulatory network. We have found that the actin assem-

bly inhibitors and depolymerization inducers cytochalasin

A and latrunculin B affected the organization of V-ATPase-

decorated vesicles in a similar manner as observed for the

loss of CAP in CAP bsr, suggesting a requirement of the

actin regulatory network and making it likely that the pri-

mary function of CAP in these processes is through its

role in regulation of the actin dynamics.

Materials and Methods

Strains and growth conditionsDictyostelium discoideum wild-type strain AX2, the CAP-deficient mutant

CAP bsr and the transformants were cultured as described (18). For rescue

experiments, CAP bsr cells were transformed with vectors allowing for the

expression of GFP fusions of CAP under the control of the actin15 gene

promoter and the actin8 gene terminator. The construction of the GFP-CAP

fusions was described previously (18,19). The GFP fusion proteins

expressed in the transformants varied in their amounts with levels nearly

identical to wild-type protein level up to twofold higher amounts. Only cells

expressing moderate amounts were chosen for the analysis. A genomic

DNA fragment of 1713 bp coding for NrampB/Nramp1 was cloned into

pDEX27 to produce an NrampB/Nramp1–GFP fusion. The vatB cDNA

was cloned into pDEX79 resulting in the expression of GFP-vatB. AX2 or

CAP bsr transformants expressing GFP fusion proteins of vatB or Nramp1

were analyzed for the presence of the fusion proteins by Western blots

using CAP domain-specific mAbs (17) or GFP-specific mAb K3-184-2 (19)

and by fluorescence microscopy.

Yeast two-hybrid assayThe yeast strain PJ69 4a was a kind gift from Dr Jurgen Dohmen

(University of Cologne). Strain Y190 and plasmids pGBKT7, pGADT7,

pAS2 and pACT2 were obtained from BD Biosciences (Palo Alto, CA).

cDNA fragments comprising the complete Dictyostelium CAP and the

vacuolar ATPase d subunit were amplified by polymerase chain reaction

(PCR) with the primers: 5´-CGGGATCCCGATGTCAGAAGCAACTATTGT-3´

and 5´-CGGGATCCCGTTAAATATGTGAAGTTGATTCA-3´ with a BamH1

linker at either flanking ends, and 5´-GGATCCCGATGGGTTTATTTG

GTGGTAGAAAACATGGTG-3´ and 5´-CTCGAGTTAAAAGATTGGAATTAT

TGATTCTTTTTG-3´ with BamHI and XhoI linker, respectively. Cyclase-

associated protein and N-CAP, a fragment of 663 bp obtained as

described (18), were, respectively, cloned in the GAL4 DNA-binding

domain vectors pGBKT7 and pAS2 using BamHI linker for CAP and

NdeI-BamHI-cloning sites for N-CAP. The V-ATPase d subunit was cloned

into the GAL4 activation domain vector pGADT7 using BamHI and XhoI

linker. A Dictyostelium library of cDNAs pooled from vegetative amoebae

was cloned into the pACT2 vector using the EcoRI-XhoI restriction sites

(43). Yeast transformations, library screening and X-Gal tests were as

described (44).

Immunoprecipitation assayFor analyzing the in vivo interactions, AX2 cells or cells expressing GFP-

CAP were axenically grown, harvested and washed twice with Soerensen

phosphate buffer (pH 6.0) and suspended in twice the volume of immuno-

precipitation (IP) buffer (PB; pH 7.4, 2 mM benzamidine, 4 mM DTT, 2 mM

EDTA and 0.5 mM PMSF). Cells were then lysed by sonication and the

solutions were adjusted to 1 � IP buffer in the presence of 0.5% Triton-X-

100 or 1% NP-40 (Fluka, Munich, Germany). The cell lysate prior to IP was

precleared with protein A Sepharose beads (Amersham Biosciences,

Freiburg, Germany) for 30 min, and the cell debris and proteins bound

nonspecifically were removed by centrifugation (2000 � g for 3 min,

4 �C). The lysate was incubated with GFP mAb K3-184-2 or CAP mAb

230-18-8 bound to protein A Sepharose beads for 2 h at 4 �C, then cen-

trifuged as above and washed several times with IP buffer. The immuno-

complexes were electrophoresed on SDS–PAGE (10–15%) and stained

with Coomassie Blue or processed for immunoblotting and probed either

with vatA or CAP-specific mAbs. The immunoprecipitated bands were

excised and analyzed by MALDI-MS (matrix-associated laser desorption-

ionization mass spectrometry) in the central facilities of the Center of

Molecular Medicine Cologne (CMMC).

Immunofluorescence microscopy and live-cell

imagingAxenically grown cells were harvested and allowed to adhere onto 18 mm

glass coverslips for 30 min, fixed with methanol for 10 min (�20 �C) or

picric acid/paraformaldehyde and processed as described by Weiner et al.

(45). Alternatively, cells on coverslips were quick frozen by floating the

coverslips on liquid N2 for 20 s and then immediately submerging them in

cold MeOH (�20 �C). For studies of cytoskeletal rearrangements, the cells

were incubated with 20 mM cytochalasin A or 1 mM latrunculin B (Sigma-

Aldrich, Munich, Germany) in 17 mM Soerensen phosphate buffer, pH 6.0,

for the desired period of time prior to fixation. For examining the sensitivity

to hypo-osmotic conditions, cells were either treated with medium or

water for 1 h, and images were obtained using a phase-contrast micro-

scope at 40� magnification or alternatively cells were fixed and stained for

vatA. Cyclase-associated protein was detected using mAb 230-18-8, the

V-ATPase was stained with mAb 221-35-2 for the A subunit (22), GFP using

mAb K3-184-2, postlysosomal compartments were detected using vacuo-

lin A-specific mAb 221-1-1 (46) and actin was labeled with mAb act-1–7 (47)

followed by incubation with Cy3-labeled goat anti-mouse IgG secondary

antibody. In paraformaldehyde-fixed cells, actin was stained with FITC-

phallodin (Sigma). Confocal microscopy was done as described (43). The

confocal images obtained at different focal planes were assembled at

equal and optimized averaging and a sectioning of 200 nm. The three

dimensional reconstitution of the images was done using the 3D option

in the Leica confocal software.

To monitor the alterations in the distribution of the CVS system, we labeled

cells with the styryl dye FM 4–64 (Molecular Probes, Karlsruhe, Germany)

(1 mg/mL) in Soerensen phosphate buffer, and time-lapse video micro-

scopy was performed according to Heuser et al. (25). To analyze the live

dynamics of GFP-CAP and Nramp1-GFP, 2–3 � 106 cells/mL were washed

and resuspended in Soerensen phosphate buffer, transferred onto a cover-

slip and allowed to adhere for 15 min. Live dynamics of GFP-CAP were

performed either with ethanol (control) or CMA (10 mM). Images were

obtained every 5 s and were processed using the accompanying Leica

software.

Determination of the endo-lysosomal pHThe endosomal pH was measured by a dual excitation ratio method (exci-

tation at 450 and 495 nm and emission at 520 nm) using FITC-dextran as a

pH probe as described by Temesvari et al. (26). Briefly, cells were grown to

2–5 � 105 cells/mL, harvested and resuspended at a concentration of

3 � 106 cells/mL in fresh axenic medium and loaded with FITC-dextran

(2 mg/mL) (70 000 Mr, Sigma-Aldrich). To monitor changes of endosomal

pH during transit of the fluid phase marker, we pulsed cells for 10 min,

washed and resuspended them in fresh nutrient medium. Basal endoso-

mal pH was measured after loading for 3 h, the time period shown to be

sufficient for the complete loading of all the endo-lysosomal compartments

with a fluid phase marker (23). The cells were then either treated with

CMA (10 mM) (Fluka) or ethanol, the CMA diluent, as control. At various

time points, cells were collected by centrifugation, washed and resus-

pended in 50 mM MES buffer, pH 6.5, and the fluorescence intensity

was measured using a PTI fluorimeter.

Sultana et al.

944 Traffic 2005; 6: 930–946

Miscellaneous methodsStandard molecular biology methods were done as described by Sambrook

et al. (48). SDS–PAGE was performed according to the method of

Laemmli (49) and immunoblotting as described in Towbin et al. (50). The

V-ATPase subunit vatA was detected with mAb 221-35-2 and comitin

(loading control) with mAb 190-68 (22,45) and the osmoregulation assay

was done as described in Gerald et al. (51).

Acknowledgments

We thank Dr S. Muller (CMMC) for performing MALDI-MS analysis,

Dr M. Maniak and B. Gassen for providing monoclonal antibodies and

R. Muller for help with IP experiments. This work was supported by grants

from the DFG (NO 113/7-3, RI 1034/2 and SFB 413), the FCI (Fonds der

Chemischen Industrie) and Koln Fortune.

References

1. Engqvist-Goldstein AE, Drubin DG. Actin assembly and endocytosis:

from yeast to mammals. Annu Rev Cell Dev Biol 2003;19:287–332.

2. Geli MI, Riezman H. Endocytic internalization in yeast and animal cells:

similar and different. J Cell Sci 1998;111:1031–1037.

3. Holtzman DA, Yang S, Drubin DG. Synthetic-lethal interactions identify

two novel genes, SLA1 and SLA2 that control membrane cytoskeleton

assembly in Saccharomyces cerevisiae. J Cell Biol 1993;122:635–644.

4. Raths S, Rohrer J, Crausaz F, Riezman H. end3 and end4: two mutants

defective in receptor-mediated and fluid-phase endocytosis in

Saccharomyces cerevisiae. J Cell Biol 1993;120:55–65.

5. Kaksonen M, Sun Y, Drubin DG. A pathway for association of receptors,

adaptors, and actin during endocytic internalization. Cell 2003;115:475–487.

6. Engqvist-Goldstein AE, Warren RA, Kessels MM, Keen JH, Heuser J,

Drubin DG. The actin-binding protein Hip1R associates with clathrin

during early stages of endocytosis and promotes clathrin assembly in

vitro. J Cell Biol 2001;15:1209–1223.

7. Engqvist-Goldstein AE, Zhang CX, Carreno S, Barroso C, Heuser JE,

Drubin DG. RNAi-mediated Hip1R silencing results in stable associa-

tion between the endocytic machinery and the actin assembly machin-

ery. Mol Biol Cell 2004;15:1666–1679.

8. Kessels MM, Engqvist-Goldstein AE, Drubin DG, Qualmann B.

Mammalian Abp1, a signal-responsive F-actin-binding protein, links

the actin cytoskeleton to endocytosis via the GTPase dynamin. J Cell

Biol 2000;153:351–366.

9. Takei K, Slepnev VI, Haucke V, De Camilli P. Functional partnership

between amphiphysin and dynamin in clathrin-mediated endocytosis.

Nat Cell Biol 1999;1:33–39.

10. Hubberstey AV, Mottillo EP. Cyclase-associated proteins: CAPacity for

linking signal transduction and actin polymerization. FASEB J

2002;16:487–499.

11. Balcer HI, Goodman AL, Rodal AA, Smith E, Kugler J, Heuser JE,

Goode BL. Coordinated regulation of actin filament turnover by a

high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1.

Curr Biol 2003;13:2159–2169.

12. Mattila PK, Quintero-Monzon O, Kugler J, Moseley JB, Almo SC,

Lappalainen P, Goode BL. A high-affinity interaction with ADP-actin

monomers underlies the mechanism and in vivo function of Srv2/

cyclase-associated protein. Mol Biol Cell 2004;15:5158–5171.

13. Bertling E, Hotulainen P, Mattila PK, Matilainen T, Salminen M,

Lappalainen P. Cyclase-associated protein 1 (CAP1) promotes cofilin-

induced actin dynamics in mammalian nonmuscle cells. Mol Biol Cell

2004;15:2324–2334.

14. Drees B, Friederich E, Fradelizi J, Louvard D, BeckerleMC, Golsteyn RM.

Characterization of the interaction between zyxin and members of the

Ena/vasodilator-stimulated phosphoprotein family of proteins. J Biol

Chem 2000;275:22503–22511.

15. Moriyama K, Yahara I. Human CAP1 is a key factor in the recycling of

cofilin and actin for rapid actin turnover. J Cell Sci 2002;115:1591–1601.

16. Goldschmidt-Clermont PJ, Janmey PA. Profilin, a weak CAP for actin

and RAS. Cell 1991;66:419–421.

17. Gottwald U, Brokamp R, Karakesisoglou I, Schleicher M, Noegel AA.

Identification of a cyclase-associated protein (CAP) homologue in

Dictyostelium discoideum and characterization of its interaction with

actin. Mol Biol Cell 1996;7:261–272.

18. Noegel AA, Rivero F, Albrecht R, Janssen KP, Kohler J, Parent CA,

Schleicher M. Assessing the role of the ASP56/CAP homologue of

Dictyostelium discoideum and the requirements for subcellular local-

ization. J Cell Sci 1999;112:3195–3203.

19. Noegel AA, Blau-Wasser R, Sultana H, Muller R, Israel L, Schleicher M,

Patel H, Weijer CJ. The cyclase-associated protein CAP as regulator of

cell polarity and cAMP signaling in Dictyostelium. Mol Biol Cell

2004;15:934–945.

20. Nishi T, Forgac M. The vacuolar (Hþ)-ATPases-nature’s most versatile

proton pumps. Nat Rev Mol Cell Biol 2002;3:94–103.

21. Patterson DJ. Contractile vacuoles and associated structures: their

organization and function. Biol Rev 1980;55:1–46.

22. Jenne N, Rauchenberger R, Hacker U, Kast T, Maniak M. Targeted

gene disruption reveals a role for vacuolin B in the late endocytic

pathway and exocytosis. J Cell Sci 1998;111:61–70.

23. Rodriguez-Paris JM, Nolta KV, Steck TL. Characterization of lysosomes

isolated from Dictyostelium discoideum by magnetic fractionation.

J Biol Chem 1993;268:9110–9116.

24. Maniak M. Green fluorescent protein in the visualization of particle

uptake and fluid-phase endocytosis. Methods Enzymol 1999;302:43–50.

25. Heuser J, Zhu Q, Clarke M. Proton pumps populate the contractile

vacuoles of Dictyostelium amoebae. J Cell Biol 1993;121:1311–1327.

26. Temesvari LA, Rodriguez-Paris JM, Bush JM, Zhang L, Cardelli JA.

Involvement of the vacuolar proton-translocating ATPase in multiple

steps of the endo-lysosomal system and in the contractile vacuole

system of Dictyostelium discoideum. J Cell Sci 1996;109:1479–1495.

27. Clarke M, Kohler J, Arana Q, Liu T, Heuser J, Gerisch G. Dynamics of the

vacuolar H(þ)-ATPase in the contractile vacuole complex and the endo-

somal pathway of Dictyostelium cells. J Cell Sci 2002;115:2893–2905.

28. Gruenheid S, Pinner E, Desjardins M, Gros P. Natural resistance to

infection with intracellular pathogens: the Nramp1 protein is recruited

to the membrane of the phagosome. J Exp Med 1997;185:717–730.

29. Cooper JA. Effects of cytochalasin and phalloidin on actin. J Cell Biol

1987;105:1473–1478.

30. Finbow ME, Harrison MA. The vacuolar Hþ-ATPase: a universal proton

pump of eukaryotes. Biochem J 1997;324:697–712.

31. Lee BS, Gluck SL, Holliday LS. Interaction between vacuolar H(þ)-

ATPase and microfilaments during osteoclast activation. J Biol Chem

1999;274:29164–29171.

32. Holliday LS, LuM, Lee BS, Nelson RD, Solivan S, Zhang L, Gluck SL. The

amino-terminal domain of the B subunit of vacuolar Hþ-ATPase contains

a filamentous actin-binding site. J Biol Chem 2000;275:32331–32337.

33. Lu X, Yu H, Liu SH, Brodsky FM, Peterlin BM. Interactions between

HIV1 Nef and vacuolar ATPase facilitate the internalization of CD4.

Immunity 1998;8:647–656.

34. Mandic R, Fackler OT, Geyer M, Linnemann T, Zheng YH, Peterlin BM.

Negative factor from SIV binds to the catalytic subunit of the V-ATPase

to internalize CD4 and to increase viral infectivity. Mol Biol Cell

2001;12:463–473.

35. Zhu Q, Liu T, Clarke M. Calmodulin and the contractile vacuole com-

plex in mitotic cells of Dictyostelium discoideum. J Cell Sci

1993;104:1119–1127.

CAP Links Endocytosis to the Actin Cytoskeleton

Traffic 2005; 6: 930–946 945

36. Bush J, Nolta K, Rodriguez-Paris J, Kaufmann N, O’Halloran T, Ruscetti L,

Temesvari L, Steck T, Cardelli J. A Rab4-like GTPase in Dictyostelium

discoideum colocalizes with V-H (þ)-ATPases in reticular membranes

of the contractile vacuole complex and in lysosomes. J Cell Sci

1994;107:2801–2812.

37. Melin P, Schnurer J, Wagner EG. Disruption of the gene encoding

the V-ATPase subunit A results in inhibition of normal growth and

abolished sporulation in Aspergillus nidulans. Microbiology

2004;150:743–748.

38. Liu T, Mirschberger C, Chooback L, AranaQ, Dal-Sacco Z, MacWilliams H,

Clarke M. Altered expression of the 100-kDa subunit of the

Dictyostelium vacuolar proton pump impairs enzyme assembly,

endocytic function and cytosolic pH regulation. J Cell Sci

2002;115:1907–1918.

39. Demaurex N. pH homeostasis of cellular organelles. News Physiol Sci

2002;17:1–5.

40. Brazill DT, Meyer LR, Hatton RD, Brock DA, Gomer RH. ABC trans-

porters required for endocytosis and endosomal pH regulation in

Dictyostelium. J Cell Sci 2001;114:3923–3932.

41. Canonne-Hergaux F, Gruenheid S, Govoni G, Gros P. The Nramp1

protein and its role in resistance to infection and macrophage function.

Proc Assoc Am Physicians 1999;111:283–289.

42. Adessi C, Chapel A, Vincon M, Rabilloud T, Klein G, Satre M, Garin J.

Identification of major proteins associated with Dictyostelium discoi-

deum endocytic vesicles. J Cell Sci 1995;108:3331–3337.

43. Knuth M, Khaire N, Kuspa A, Lu SJ, Schleicher M, Noegel AA. A novel

partner for Dictyostelium filamin is an alpha-helical developmentally

regulated protein. J Cell Sci 2004;117:5013–5022.

44. Gietz RD, Schiestl RH, Willems AR, Woods RA. Studies on the trans-

formation of intact yeast cells by the LiAc/SS-DNA/PEG procedure.

Yeast 1995;11:355–360.

45. Weiner OH, Murphy J, Griffiths G, Schleicher M, Noegel AA. The actin-

binding protein comitin (p24) is a component of the Golgi apparatus.

J Cell Biol 1993;123:23–34.

46. Rauchenberger R, Hacker U, Murphy J, Niewohner J, Maniak M.

Coronin and vacuolin identify consecutive stages of a late, actin-coated

endocytic compartment in Dictyostelium. Curr Biol 1997;7:215–218.

47. Simpson PA, Spudich JA, Parham P. Monoclonal antibodies prepared

against Dictyostelium actin: characterization and interactions with

actin. J Cell Biol 1984;99:287–295.

48. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. Cold Spring

Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.

49. Laemmli UK. Cleavage of structural proteins during assembly of the

head of bacteriophage T4. Nature 1970;227:680–685.

50. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins

from polyacrylamide gels to nitrocellulose sheets: procedure and some

applications. Proc Natl Acad Sci USA 1979;76:4350–4354.

51. Gerald NJ, Siano M, De Lozanne A. The Dictyostelium LvsA protein is

localized on the contractile vacuole and is required for osmoregulation.

Traffic 2002;3:50–60.

Sultana et al.

946 Traffic 2005; 6: 930–946